PER2) has a role in the intracellular signaling pathways of long-term potentiation and has implications for synaptic plasticity. We aimed to assess the association of PER2 C111G polymorphism with cognitive functions in subjective cognitive decline (SCD). Methods: Forty-five SCD patients were included in this study. All participants underwent extensive neuropsychological investigation, analysis of apolipoprotein E (APOE) and PER2 genotypes, and neuropsychological follow-up every 12 or 24 months for a mean time of 9.87 ± 4.38 years. Results: Nine out of 45 patients (20%) were heterozygous carriers of the PER2 C111G polymorphism (G carriers), while 36 patients (80%) were not carriers of the G allele (G non-carriers). At baseline, G carriers had a higher language composite score compared to G non-carriers. During follow-up, 15 (34.88%) patients progressed to mild cognitive impairment (MCI). In this group, we found a significant interaction between PER2 G allele and follow-up time, as carriers of G allele showed greater worsening of executive function, visual-spatial ability, and language composite scores compared to G non-carriers. Conclusions: PER2 C111G polymorphism is associated with better language performance in SCD patients. Nevertheless, as patients progress to MCI, G allele carriers showed a greater worsening in cognitive performance compared to G non-carriers. The effect of PER2 C111G polymorphism depends on the global cognitive status of patients. Background: Periodic circadian protein homolog 2 ( Subjective cognitive decline (SCD) is defined as a self-experienced decline in cognitive capacity during which individuals have normal performance on standardized cognitive tests . Recent PER2), a clock-controlled gene, has a specific role in the intracellular signaling pathways of long-term potentiation (LTP) and has implications for synaptic plasticity and learned behaviors ) were carriers of the CG genotype (G carriers), while 36 patients (80.00% [95% CI 68.04–91.96%]) were carriers of the CC genotype (G non-carriers). None of the patients was carrier of the GG genotype. The genotypic distribution of PER2 gene was in Hardy–Weinberg equilibrium . We found no difference between G carriers and G non-carriers with respect to age at baseline, disease duration , sex, score of scale for depression, APOE ε4 allele frequency, Mini-Mental State Examination (MMSE). G carriers had less years of education  as compared to G non-carriers  were carriers of the carriers . No diffcarriers .z = 2.33, p = 0.018). No difference with respect to the other cognitive domains were found between G carriers and G non-carriers . The model explained 52.7% (Nagelkerke R2) of the variance in conversion and correctly classified 88.6% of cases. Of the covariates considered for the analysis, only lower education  and higher language score  were significantly associated with a higher proportion of G allele  patients progressed to MCI (pSCD). Thirty patients (65.12% [95% CI 50.87–79.37]) did not progress. Two out of 15 pSCD patients (13.33%) and five out of 30 npSCD patients (16.67%) were G carriers. There were no differences between pSCD and npSCD groups with respect to age at baseline, disease duration, follow-up time, years of education, MMSE, Hamilton Depression rating scale (HDRS), MAC-Q, and aluation .PER2 polymorphism on changes on cognitive performance during follow-up, we ran generalized estimating equations. We considered baseline evaluation and last follow-up neuropsychological evaluation as within-subjects variables, composite z-scores of each cognitive domain as dependent variables, and PER2 genotype (G carrier or G non-carriers) and follow-up time  as predictors. The model included follow-up time and interaction of follow-up time with PER2 genotype.In order to explore the effect of PER2 polymorphism on changes in cognitive performance during the follow-up . This provides useful data to exclude possible confounding factors and to support our findings. Finally, our longitudinal analyses are based on data from a very long follow-up time.This study has remarkable strengths. First of all, we would like to emphasize that this is the first study assessing the effect of SD value of this variable. Another limitation is the lack of AD biomarkers data. Finally, as it is a single-center study, there may be biases with regard to assessment and diagnosis procedures.The small size of our cohort of patients is the first limitation of our study. In particular, only nine patients were G carriers in the whole sample, and only two patients were G carriers in the pSCD group. This limits our conclusions and means that, in order to ground our findings, we shall expand our sample. Also, we must point out that, despite a long mean follow-up time, we encountered a high variability in follow-up times, as shown by the PER2 C111G polymorphism differently influences cognitive functions according to the global cognitive status of the subject. In particular, the PER2 G allele seems to provide an advantage in relation to language in cognitively healthy subjects. Nevertheless, in patients who progress to objective cognitive impairment, this advantage seems to disappear, and the G allele is associated with a greater decline in language, executive function, and visual–spatial ability. Our findings pave the way to future research on the implication of the PER2 gene on the neurobiological mechanism(s) underling cognitive reserve, as well as the neuropsychological functions in healthy and in cognitively impaired patients and in neurodegenerative diseases.


Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall. Human serum transferrin (Tf), also known as serotransferrin, is a ~80 kDa glycoprotein that transits blood plasma, whose function is to maintain iron homeostasis by transporting iron from its absorption site to every cell through the blood vessels. Its structure consists of two homologous lobes, an N-terminal lobe and a C-terminal lobe, each subdivided into two domains, in between which a metal-binding site is found, thus allowing Tf to bind two ferric ions per molecule ,2. The tTransferrin’s mechanism of iron transport draws special attention because it occurs in endothelial cells of the blood–brain barrier. These tightly bound cells restrict the influx of material from the bloodstream, limiting the possibilities of treatment for various neurological diseases; however, transferrin is able to cross this barrier thanks to receptor-mediated endocytosis ,6. MoreoAlthough the behavior of a macromolecule in its physiological environment is not the same as it is in vitro, it is helpful to know about it to fine-tune experimental setups. The aim of this contribution is to provide insight into the structural complexity of native human serum transferrin and the challenges that go along with it. When crystallizing transferrin, two important factors must be considered: its great conformational flexibility and the fact that its mechanism of iron release is pH-dependent ,3,6,15 aNumerous crystals of apo- and holo-Tf have been grown by the classic vapor-diffusion technique, showing great optical properties and well-defined morphology , but limAfter exploring a plethora of strategies, a crystallographic model of apo-Tf has been constructed from X-ray diffraction data from one of the best crystals grown under the conventional vapor-diffusion technique. The data collection and refinement statistics for our apo-Tf model, deposited under the 7Q1L PDB code, are summarized in The data for our apo-Tf model are good enough for visualizing the general protein structure, allowing the location of two NAG moieties bound to residue Asn413. Some extra density could be observed after the second NAG element, which points to a potential elongation of the glycosyl chain that could be the key factor hampering crystal quality improvement. As has been reviewed, glycosylation does not necessarily prevent crystallization—it may even help—but the resolution limit and diffraction quality may suffer . Superpog) calculated from the slope of the Guinier plot described as ln(Is) vs. s2, where s = 4 πsin(θ)/λ is the scattering vector (2θ is the scattering angle and λ is the wavelength) [g into Guiner zone (1.0-1.3). The linearity of the Guinier plot is considered a quality measurement of the data, but it does not ensure the ideality of the sample. Guinier plots for apo-Tf and holo-Tf were linear  . For gloe linear , and theg, Dmax, Kratky plot, and Porod coefficient, it is possible to see that holo-Tf is more elongated and more flexible, and this is more evident at low pH.The dimensionless Kratky plot was used to investigate the flexibility (and shape) of the proteins B. The mamax corresponds to the smoothest and most positive distribution. Differences in the Dmax of a protein relate to conformational changes. Additionally, it is possible to calculate the Rg from the Pair-Distance Distribution Function and compare its value with that estimated from the Guinier plot. In the case of holo-Tf, differences in Dmax were observed as the pH value lowered, suggesting a conformational change induced by an acidic environment  can be obtained from analysis of the SAXS data by means of the Pair-Distance Distribution Function (P(r)) C, which ironment . For apoironment C, as welx values , showed g and Dmax), for pH 5.5. For simplification, the last case (holo-Tf at pH 5.5) is shown in 2 is decreased from 33.5 to 1.74) holo-Tf model by SREFLEX.For all the data sets, SAXS modeling was performed by using an ab initio reconstruction of a protein structure by simulated annealing using a single-phase dummy atom . For apo2 value (better fitting). The same is true from State 2 to State 3. Therefore, the higher the number of states, the higher the flexibility of the model. In 2 fitting value. In To further investigate the pH dependence of transferrin, we employed a SAXS-based pseudo-atomic modeling approach using the MultiFoXS server . This teg and Dmax values for the three-state model at all pH values . After elution, apo- and holo-Tf were dialyzed against 15 mM HEPES pH 8.0 with 20 mM NaHCO3 and 50 mM NaCl using a 6–8 kDa MWCO membrane, at 4 °C for 2 h, then other 2 h with a fresh buffer, and finally overnight with a fresh buffer. Finally, apo and holo were concentrated by centrifugation in Amicon tubes at 3000× g in 15-min cycles at 4 °C.Human serum transferrin was commercially obtained  in both its iron-free (apo) and iron-loaded (holo) forms. The same conditions were used for purifying both. The proteins were suspended in a buffer containing 50 mM TRIS-HCl pH 8.0 with 20 mM NaHCO−1 using Index screen #88  condition as the precipitant solution, which consists of 0.2 M ammonium citrate tribasic, pH 7, and 20% PEG 3350. Crystals were grown at 18 °C by the vapor-diffusion method as previously standardized [Transferrin crystals were grown at a stock concentration of 20 mg mLdardized ,30. Prior to data collection, crystals were subject to cryo-preservation by being flash cooled in liquid nitrogen and then stored. Because crystal quality was the main issue, several protocols were assayed, including the use of several cryoprotectants  at different concentrations and soaking times, the use of naked crystals, or long soaking (24 h) for crystals obtained in capillaries by the counter-diffusion technique. The crystal quality of a total of 103 different samples was evaluated from X-ray diffraction data collected from beamlines XALOC of ALBA , ID-30B, ID23-1, ID30A-3 of ESRF , as well as from SSRL ,32 synchv/v) glycerol and immediately put into the X-ray beam under cryogenic conditions at 100 K. The wavelength of incident X-rays was 1.13 Å with an Eiger 16M detector. Data-collection strategies included high-redundancy data, and each sample was rotated in 0.25° increments. The HKL3000 [The X-ray data collection was performed at the microfocus beamline BL-14-1 at the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory . The crystals were cryo-protected with 30% , for both Tf-apo and Tf-holo samples, was measured. The stock protein sample was at a concentration of 20 mg mL−1 and it was diluted up to 5 mg mL−1 and 2.5 mg mL−1, using the buffer corrected at pH 7.0 and 5.5 for the tests on these pH. We did not check the final pH of the solution, but we calculate that during the dilution, the pH should not be significantly different from the expected value. Since the scattering curves for Tf did not display any concentration dependence at the highest concentrations (−1) was used in our analysis. Merging, subtracting, and subsequent analysis were performed using PRIMUS [SAXS experiments for transferrin were carried out at BM29 ,43 beaml handler  in flow- handler . Before trations , this cog PRIMUS  and Scatg PRIMUS  softwareg PRIMUS , SREFLEXg PRIMUS , and Mulg PRIMUS  softwareg PRIMUS . Modeling PRIMUS .The Small-Angle X-ray Scattering data clearly showed a strong pH dependency and open-structured conformation at different times and pH values. Static snapshots that we attempted to derive from X-ray diffraction data were hampered by intrinsically low crystal quality and/or high sensitivity to crystal manipulation. We can conclude that the glycosylation is perhaps the main reason that all the efforts to improve crystal quality were not successful, along with the high flexibility of the structure, as shown in the SAXS experiments in the solution. From this study, we can propose conformational details of the mechanism of pH-dependent iron release from transferrin, including a monoferric intermediate state, which will have important biomedical research impacts on the transport of iron or ligands against brain diseases.


P ＜ 0.001, OR = 1.530; P ＜ 0.001, OR = 1.485)，serum bilirubin  and international normalised ratio (INR)  were predictors of both the 30-day and 4-year mortality rates. Renal failure  performed good in prediction of the 4-year mortality. The mortality of fungaemia is high. Age，the serum bilirubin and INR are good predictors of the 30-day and 4-year mortality rates of fungaemia. Renal failure has good performance in predicting the long-term mortality.Bloodstream fungal infections have a high mortality rate. There is little data about the long-term mortality rate of fungaemia.This study aimed to explore the mortality of fungaemia and the influencing factors associated with death. In total, 204 intensive care unit (ICU) patients with fungaemia from Multi-parameter Intelligent Monitoring in Intensive Care-III (MIMIC-III) Database were studied. Age, gender, major underlying diseases, data about vital signs and blood test results were analysed to identify the predictors of the mortality and prognosis of fungaemia in ICU patients. Cox regression models were constructed, together with Kaplan−Meier survival curves. The 30-day, 1-year, 2-year, 3-year and 4-year mortality rates were 41.2%, 62.3%, 68.1%, 72.5% and 75%, respectively. Age ( Fungaemia has high short-term and long-term mortality rates. An Israel study conducted in the early 1990s reported an one-month mortality of 42% and a four-year mortality of 86% , 4, broaThe number of antifungal treatment options has increased since the approval of caspofungin by Food and Drug Administration (FDA) in 2001 . HoweverThis retrospective study was based on MIMIC-III Database . The inc2）on the day of blood culture test. Severity of fungaemia was estimated using the sequential organ failure assessment (SOFA) score on the day of blood culture test. The patients'comorbidity scores were evaluated with the Elixhauser_vanwalraven scoring system [The variables extracted from MIMIC-III Database included age on the day of blood culture test, gender and major underlying diseases, such as diabetes mellitus(diabetes), congestive heart failure, chronic obstructive pulmonary disease (COPD), renal failure, liver disease and solid tumours. The variables of vital signs were mean blood pressure  on the day of blood culture test. Other variables of blood test results were serum bilirubin level , serum creatinine level , blood urea nitrogen , INR and pulse oxygen saturation(SPOg system . The worn), and the intergroup differences were analysed with Chi-square test or Fisher's exact test. The Shapiro−Wilk normality test showed that some data did not conform to a normal distribution (P < 0.05). Therefore, the measurement data in this study were described by P50  and a non-parametric rank-sum test was used for intergroup comparison. The factors associated both 30-days and the 4-year mortality were identified. The factors whose P values were ⩽0.1 in univariate analyses were entered into Cox regression model in the way of forward selection  until the final model was achieved. Kaplan–Meier survival curves for significant predictors of the 4-year mortality were constructed and analysed using the log-rank test. All analyses were performed using SPSS (IBM SPSS Statistics 25).The count data were represented by case number  with fungaemia who met the inclusion criteria were analysed, and their 225 fungaemia episodes were reviewed. The patients with fungaemia had a median age of 64.7 years (interquartile range: 51.4–74.6) and 57.8% of them were male. The 30-day and 4-year mortality rates were 41.2% and 75%, respectively, while the rest of the population of the database were 13.6% and 34.7%, respectively, as shown in As shown in P = 0.001, OR = 2.359 (1.433–3.882)), but also in predicting 4-year mortality ). Therefore, INR is a good predictor of mortality in fungaemia. In other reports, INR was associated with in-hospital mortality [In this study, results show that age，INR and bilirubin are good predictors of short-term and long-term mortality of fungaemia. In particular, the INR is statistically significant in predicting 30-day mortality (ortality , 13. Oneet al. Pierrakos et al. [Bilirubin can be used as an independent predictor of the short-term and long-term prognosis of fungaemia. Bilirubin is the principal tetrapyrrole, bile pigment and catabolite of haem . It inhis et al.  found ths et al. . It is det al. [Bilirubin, as a biomarker for the prevalence of chronic diseases and prediction of all-cause mortality , is alsoet al.  reportedet al.  was usedet al.'s study, which found that renal failure was associated with cardiovascular death in patients with heart failure. This is because fungaemia aggravates chronic inflammation of the kidney, which increases the risk of death [Renal failure could be used as an independent predictor of the long-term prognosis of fungaemia. Similar results have been found in other disease studies, such as Toshitaka Okabe  et al.'sof death .In this study, nine cases of patients with fungaemia accompanied by solid tumour, only one died within 30 days. The 30-day mortality rate of fungaemia patients with solid tumour was reduced. Chen  et al. ret al. [et al. [In addition, we have illustrated that the 1-month, 1-year, 2-year, 3-year and 4-year mortality rates of fungaemia are 41.2%, 62.3%, 68.1%, 72.5% and 75.0% among ICU patients in this study, respectively. Siriluck Anunnatsiri et al.  reportedSaccharomyces cerevisiae infection. General patient information is, male, 50.8 years old, with renal failure and liver disease, long-term use of glucocorticoids and immunosuppressants. After diagnosis of fungaemia, the survival period was 218 days. S. cerevisiae is widely used in baking and brewing industry, which is a very rare infection in human beings. Patricia Muñoz [S. cerevisiae should be carefully reassessed, especially in immunosuppressed or critically ill patients. Davide Fiore Bavaro reported that targeted therapy might enhance or inhibit immunity in patients with autoimmune idiopathic diseases. However, the drugs would have some adverse reactions, such as opportunistic infections [Among the fungal strains in this study, there is a case of fungaemia caused by ia Muñoz  suggestefections .This study has six limitations: ① the sample size was small because the data were from a single centre and the incidence of fungaemia was low, which might lead to non-universal findings. ② Fungaemia are often ‘opportunistic’ infections, caused by important antibiotic selective pressure, parenteral nutrition and major abdominal surgery. However, this study is a retrospective study rather than a prospective study, which inevitably leads to the limitation. ③ There were no data control group in this study. ④ The study did not stratify outcomes according to therapeutic interventions. ⑤ Polymicrobic infections were not used as a prediction; ⑥ The long-term and short-term mortality of fungaemia might be different in different areas because of differences in healthcare quality, economic development and culture.In conclusion, the incidence rate of fungaemia is 0.53% among ICU patients. There is an appreciable and gradual increase in mortality after an episode of bloodstream fungal infection. The SOFA score and Elixhauser vanwalraven score have good performance in predicting the 30-day mortality. Treatment of organ dysfunction and control of underlying diseases may help improve the short-term prognosis of fungaemia. Age，INR and bilirubin are good predictors of the short-term and long-term mortality rates of fungaemia. Renal failure has good performance in predicting the long-term mortality.


The optimized nanoparticulate system is speculated to be a successful approach for boosting the effect of BER in treating CNS diseases, such as Alzheimer’s disease, through intranasal therapy.This research aimed to design, optimize, and evaluate berberine-laden nanostructured lipid carriers overlaid with chitosan (BER-CTS-NLCs) for efficient brain delivery via the intranasal route. The nanostructured lipid carriers containing berberine (BER-NLCs) were formulated via hot homogenization and ultrasonication strategy and optimized for the influence of a variety of causal variables, including the amount of glycerol monostearate (solid lipid), poloxamer 407 (surfactant) concentration, and oleic acid (liquid lipid) amount, on size of the particles, entrapment, and the total drug release after 24 h. The optimal BER-NLCs formulation was then coated with chitosan. Their diameter, in vitro release, surface charge, morphology, ex vivo permeability, pH, histological, and in vivo (pharmacokinetics and brain uptake) parameters were estimated. BER-CTS-NLCs had a size of 180.9 ± 4.3 nm, sustained-release properties, positive surface charge of 36.8 mV, and augmented ex-vivo permeation via nasal mucosa. The histopathological assessment revealed that the BER-CTS-NLCs system is safe for nasal delivery. Pharmacokinetic and brain accumulation experiments showed that animals treated intranasally with BER-CTS-NLCs had substantially greater drug levels in the brain. The ratios of BER brain/blood levels at 30 min, AUC Natural products have recently acquired much attention because of their anti-inflammatory, antiplatelet, antioxidant, and other health benefits ,2. For dIndividuals with AD are now treated with medications that raise acetylcholine levels in brain tissue, such as rivastigmine, galantamine, and donepezil . HoweverDrug delivery systems based on nanotechnology have emerged as potential vehicles for encapsulating, protecting, as well as delivering embedded pharmaceuticals to the desired site at a specified rate and/or extent. Novel delivery systems have been devised to improve BER’s physicochemical characteristics and brain bioavailability. Carbon nanotubes , chitosaDrugs may be transported to the brain through the nose without crossing the BBB, lowering plasma levels and decreasing side effects . The olfNumerous approaches for brain delivery have been investigated, with nano-systems emerging as the most promising. Nanostructured lipid carriers (NLCs), a new generation of lipid nanoparticles, were designed to address some of the drawbacks of solid lipid nanoparticles (SLNs). NLCs include both solid and liquid lipid matrix ; this reHowever, the duration of stay of nanoparticles in the nasal cavity is significantly short owing to mucociliary clearance, which results in insufficient drug absorption. Chitosan (CTS), a naturally occurring polymer, has recently been used in nasal delivery systems due to its mucoadhesion merit, which boosts drug penetration through the nasal mucosa and impedes mucociliary clearance as well . AdditioFabrication and evaluation of BER-loaded NLCs for the management of AD through oral administration have been described elsewhere . This stIt is essential to select suitable components during the formulation of NLCs in order to obtain optimal uniform PS, stability, and entrapment. Surfactants, for instance, significantly affect the quality and PS of NLCs. Poloxamer 407 was used in this study owing to its resistance to particle agglomeration and suppression of P-glycoproteins involved in the brain endothelial cell efflux mechanism . By expep-value < 0.05). The relations between the independent variables: GMS amount (X1), poloxamer 407 concentration (X2) and OA amount (X3), and the response variables ((Y1: PS), (Y2: EE %), and (Y3: CDR %)) was best described by the mathematical equations mentioned in ZP values of the NLC formulations ranged between −35.1 and −27.5 mV . The higAs shown in Entrapment of more than 90% was obtained when the GMS amount was more than 190 mg . The optimal NLCs are characterized by low GMS amount and high OA amount, and poloxamer 407 concentration. This combination yielded 142.1 ± 5.7 nm particles capable of holding 80.3 ± 3.1% of the added drug and releasing 85.6 ± 4.2% after 24 h. These measured values were very similar to the values predicted by the model, 119.8 nm, 76.1%, and 88.2, respectively, proving the accuracy of the optimization methods used in this analysis  than uncoated NLCs (142.1 ± 5.7), suggesting that CTS attached effectively to the NLCs’ surface, resulting in their increased size . The surThe in vitro release of BER-CTS-NLCs vs. BER-SOL in SNES is shown in 2) of BER was permeated from BER-CTS-NLCs, while 45.2 ± 3.2% (271.2 ± 19.25 µg/cm2) of BER was permeated from the BER-SOL. The findings of this experiment indicate that BER-CTS-NLCs permeated more than BER-SOL (p < 0.05)  . The perpathways , is expeThe pH of BER-CTS-NLCs must be determined to ensure that the system is safe to administer intranasally. The pH of nasal mucosa in its normal physiological state ranges between 4.5 to 6.5, while the pH of the BER-CTS-NLCs was observed to be 5.7. These observations suggest that the BER-CTS-NLCs formulation is physiologically compatible and expected to have no pH-related tissue-damaging effects.A histological assessment was performed to determine the potential toxicity of the BER-CTS-NLCs system. The photomicrographs of control rats (without any administration) A showed p < 0.05) half-life and a slower clearance rate in the brain. Between 60 and 360 min, the level of BER in the brain in the case of BER-CTS-NLCs was substantially greater (p < 0.05) than that in the case of BER-SOL (IV and IN). This may be because the BER-CTS-NLCs have a prolonged residence period in the nasal cavity, resulting in enhanced nasal uptake and the potential of continuously delivering the drug to the brain. The accumulation of the drug in the brain was statistically negligible within the first 45 min. This may be due to the time needed to transfer BER-CTS-NLCs and the drug’s delayed release from the lipid matrix. This has the benefit over traditional drug solutions in that the concentration of drugs in the brain may be maintained for a more extended period.The pharmacokinetics and brain distribution studies were conducted using male Wistar rats after IV (BER-SOL) and IN  administration. For up to 6 h, the level of BER in the brain and blood was measured . The phaIncreased BER uptake from BER-CTS-NLCs may have originated from the small PS and lipidic content of NLCs as well as from shielding against metabolic enzymes found in the nasal mucosa lining the cavity . Additiobrain/AUCblood ratios were 1.22, 0.99, and 0.24, respectively. These results indicate direct BER transport to the brain via the intranasal route and higher accumulation of BER in the brain via the nanoparticles.After 360 min, the level of BER in the brain was 13.2 and 4.4 times for the NLCs formulation more than for the BER-SOL (IV) and BER-SOL (IN), respectively. These results are in line with those of Seju et al. . The braThe DTP and DTE are used to determine the quantity of drugs that enter the brain directly through the olfactory pathway. These were determined utilizing data from the distribution to tissue/organs upon IV and IN administration. The highest DTE and DTP were seen in BER-CTS-NLCs, at 509.2 and 80.4, respectively, suggesting that CTS-NLCs formulations enhanced BER brain targeting compared to the solution.NLCs may penetrate nasal epithelial cells, according to Mistry et al. ,53. ThisBerberine chloride (BER), chitosan , poloxamer 407, glycerol monostearate (GMS) (MP: 52–54 °C and MW: 358.63), dialysis membrane (MW: 12 kDa), chloroform (HPLC grade), acetonitrile (HPLC grade), soybean lecithin (L-a phosphatidylcholine), methanol (HPLC grade), and oleic acid (OA) were procured from Sigma–Aldrich Chemical Co. . Other chemicals and reagents were of analytical grade.1), concentration of poloxamer 407 , and amount of OA  were picked as the independent variables. Each independent variable was analyzed at three levels: high, medium, and low . Three-dimensional response surface plots were made in R using the plot3D R package [NLCs were optimized in this research utilizing a Box-Behnken (BB) design. The amount of GMS  was used to detect BER quantity. A mobile phase of 0.05 mol/L NaH2PO4 and acetonitrile (70:30 v/v) (pH adjusted with phosphoric acid to 2.5) was pumped at a 1 mL/min flow rate through the HPLC system. UV detection was carried out at 345 nm at 30 °C. The injection volume and the retention time were 20 µL and 5 min, respectively. The developed HPLC assay was very sensitive and produced a linear calibration curve encompassing the concentration range 0.01–1 µg/mL (R2 = 0.999).BER quantity was estimated using a validated HPLC method . The AgiThe particle diameter and surface charge of the NLC formulations were investigated using a dynamic scattering technique  . The NLCThe entrapment of BER in NLCs formulations was estimated indirectly (based on free BER). By adding 0.1N HCL to the prepared formulations, the pH was modified to 1.2 and the nanoparticles were precipitated. The formulations were centrifuged at 11,000 rpm for 45 min at 16 °C via a cooling centrifuge  . Then, t2 diffusion surface area, were used to estimate the cumulative amount of BER released as a function of time. A certain amount of different BER-NLC formulations (3 mg BER in each) was added. Fifty milliliters of SNES  with a pH of 5.5 were poured into the receptor chamber [Vertical Franz cells, with 5 cm chamber . The SNE chamber . The recw/v) for 20 min under stirring at 25 °C and then refrigerating until further testing [The CTS coated BER-NLCs were formulated by dropping 5 mL of the optimized formulation to 5 mL of 0.5% CTS acetic acid solution ( testing .w/v) was added to the copper grid and allowed to dry at 25 °C [The size and surface charge of BER-CTS-NLCs were assessed, as previously stated. The morphological evaluation of BER-CTS-NLCs was evaluated using a TEM  . NLCs diat 25 °C .As detailed before, in vitro release experiments of BER via the dialysis membrane from BER-CTS-NLCs and BER solution (BER-SOL) were conducted using vertical Franz cells.2 was used to conduct ex vivo diffusion experiments. The butcher provided us with freshly removed sheep nasal mucosa, which was thoroughly cleaned to remove adhering tissues. The mucosa was then rinsed with phosphate buffer saline (PBS) and sank in PBS for 30 min [A Franz diffusion cell with a surface area of 2.5 cmr 30 min . A 0.2 mr 30 min  was usedAssessment of the pH of the optimized formulation is necessary to guarantee that it does not irritate the nasal tissue. The pH was evaluated by adding 10 mL of the optimized BER-CTS-NLCs in a beaker and measuring the pH via a digital pH meter .The animals employed for in vivo experiments were Wistar male rats (210–245 gm). The protocol was approved by the ethics committee of the Faculty of Pharmacy at Beni-Suef University (Acceptance No: 021–211) and the Research Ethics Committee at Taif University , which is accredited by the national committee for bioethics (No: HAO-02-T-105).Histological analysis of toxicity markers such as inflammation, abnormal symptoms, and fibrosis was used to confirm the safety of BER-CTS-NLCs intranasal administration and exclude the potential of nasal toxicity . Six malNasal Absorption and Brain Distribution Analysis of BER-CTS-NLCs.Wistar male rats were utilized in the pharmacokinetics study. The animals were kept in a normal laboratory environment . The rats were assigned to three groups: group A was given BER-SOL (IV), group B was given BER-SOL (IN), and group C was given BER-CTS-NLCs (IN). Every group was then split into six time-based subgroups of 10, 30, 60, 120, 240, and 480 min. Each subset comprised three rats; each of them got a 5 mg/kg BER dosage. In group A, BER-SOL was injected into the tail vein of rats. While in groups B and C, BER-SOL and BER-CTS-NLCs were delivered into each nostril with a micropipette attached to a low-density polyethylene tube having 0.1 mm internal diameter. Before nasal administration, the rats were anesthetized with intraperitoneal pentobarbital sodium (35–50 mg/kg). The rats were sacrificed by injecting pentobarbital sodium (overdose) at the indicated time intervals, and blood was then collected from the retino-orbital vein in EDTA coated Eppendorf tubes.Before HPLC drug analysis, the blood was fractionated at 4000 rpm for 20 min, and the supernatant was separated and stored at −21 °C. The rats were sacrificed at the exact moment the blood was collected to remove the brain. After being cleaned twice with normal saline, the brain was weighed and cleaned of adherent tissue/fluid. A cold normal saline solution was used to homogenize the brain . The brain homogenate was centrifuged at 4000 rpm , and then the supernatant was separated and stored at −21 °C.After plasma was isolated from the blood samples, the proteins were precipitated by adding an equal volume of acetonitrile. Plasma samples were then vortexed for 3 min and then centrifuged at 11,000 rpm for 25 min. 20 μL of the supernatant was injected into the HPLC apparatus. For the brain homogenates, 100–500 µL were extracted with acetonitrile (1:1) and then vortexed for 2 min ; 20 μL of the supernatant was subsequently run through the HPLC system.BER brain and plasma concentrations after IN and IV were analyzed employing the pharmacokinetic software . Additionally, metrics such as AUC, Cmax, and Tmax were computed. The profiles were utilized to directly determine the BER maximum concentration and time required to reach that concentration. The AUC was calculated up to 480 min using the linear trapezoidal method. The drug targeting efficiency (DTE), defined as the time-weighted average partitioning ratio, and the direct transport percentage (DTP), defined as the direct drug transfer from the nose to the brain, were measured as previously reported .p-value less than 0.05 was considered significant.Each experiment was performed three times. Statistical differences between groups were evaluated where appropriate using one-way ANOVA with Tukey post hoc test, as incorporated in the aov function in R software . Throughout the research, a brain/AUCblood ratio, DTP, and DTE values than BER-SOL (IN), suggesting that CTS-NLCs may be used to target the brain through the intranasal route. Lastly, according to the results of this research, BER-CTS-NLCs may be a successful approach for boosting the effect of BER in treating CNS diseases such as AD through intranasal therapy. However, substantial clinical data are required to assess the formulation’s efficacy and risk/benefit ratio in humans.BER-CTS-NLCs were successfully designed, optimized, and evaluated for brain targeting. All criteria determined were within acceptable ranges, including the size of particles, surface charge, and pH. BER-CTS-NLCs exhibited prolonged drug release behavior and boosted drug penetration across sheep nasal mucosa. Besides, histopathological assessment indicated that the BER-CTS-NLCs system is safe for nasal delivery. Pharmacokinetic and brain targeting studies showed that BER-CTS-NLCs (IN) had greater drug concentrations in the brain, AUC


The Fourier transform infrared (IR) and X-ray powder diffraction (XRD) analyses confirmed that the poly(St-co-MMA)/FA composites were successfully prepared. The morphology analysis indicated that the composite microspheres show good sphericity, and FA powder was evenly dispersed in the matrix. The apparent density of the microspheres was between 1 and 1.3 g/cm3, which is suitable for hydraulic fracturing. Furthermore, the compressive strength and thermostability were dramatically improved with the incorporation of FA, which could withstand high pressures and temperatures underground. The obtained poly(St-co-MMA)/FA composite microspheres are promising for application as an ultra-lightweight (ULW) proppant in oil or gas exploitation, which provides a new approach for the design of high performance proppants.Hydraulic fracturing is an important technology for the exploitation of unconventional oil or gas reservoirs. In order to increase the production of oil or gas, ultra-lightweight proppants with a high compressive strength are highly desirable in hydraulic fracture systems. In this work, a new type of ultra-lightweight proppant, poly(styrene- Hydraulic fracturing is widely used to increase the efficiency of oil and gas exploitation, especially in low permeability oil and gas wells . ProppanThe density of proppants depends on the composition, which can be adjusted by choosing the most suitable materials . In our In order to improve the mechanical strength of the polymers, inorganic fillers would be a good choice, as they can reinforce the mechanical strength dramatically. Fly ash (FA) is an important inorganic filler which is widely used in industrial production. Thus, the comprehensive utilization of the waste fly ash is urgent and significant to the long-term sustainable development of industrial production and environmental protection . That isco-MMA)/FA composite microspheres with an ultra-low density and outstanding mechanical and good thermal stability were prepared via in situ suspension polymerization, exhibiting promising applications as ULW proppants. This kind of proppant would not only enhance the efficiency of oil and gas production, but can also realize the value-added utilization of fly ash, thus turning waste into treasure and harm into profit.In this work, poly were purchased from Tianjin Fuchen Chemical Regents Factory . Deionized (DI) water was used throughout the experiments.Fly ash  was provided by HSBC New Materials Co., Ltd., Zhengzhou, China. Styrene monomers , divinyl benzene (DVB), and stearic acid (SA) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. . Methyl methacrylate (MMA), benzoyl peroxide (BPO), γ-aminopropyltriethoxysilane (APTES), and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. . MgClA total of 120 mL toluene was added into a 250 mL flask as dispersion medium, then a certain amount of FA and APTES were added and reacted at 90 °C by mechanical stirring for 2 h. The selected weight ratio of APTES to FA was 4:100. Then, the SA was added and dispersed in the mixture and continued to reflux at 90 °C by mechanical stirring for 2 h. The selected weight ratio of SA to FA was 4:100. The product was filtered under vacuum, washed successively with deionized water and absolute ethanol several times, then dried at 60 °C for 12 h.2·6H2O and 1.6 g of NaOH were introduced into the mixture with a vigorous stirring for 10 min. Meanwhile, 0.3 g of BPO, 3.0 g of DVB, and a certain amount of modified FA were dissolved and dispersed in 10.0 g of MMA and 10.0 g of St in a beaker. Next, the mixture was added to the flask, The stirring speed was controlled at 290 r/min and the mixture was heated to 78 °C for 1 h, then maintained at 85 °C for 2 h and 90 °C for 2 h. The product was obtained by filtration, then washed with 60 mL of deionized water and 60 mL of methanol. Finally, the obtained poly(St-co-MMA)/FA composite microspheres were dried for 12 h at 60 °C. As a contrast, the pure poly(St-co-MMA) microspheres were prepared through a similar procedure without adding modified FA. The composites microspheres with a particle size of 20–40 mesh were selected and used as the research object.A typical preparation procedure is described as follows : 0.4 g o−1 using the KBr pellet technique. The microstructure and surface morphology of these particles were obtained by a Binocular Microscope (Olympus Cx43). The thermogravimetry analysis (TGA) was conducted via a NETZSCH TG 209F3 instrument (NETZSCH Scientific Instruments Trading (Shanghai)Ltd., Shanghai, China) under N2 atmosphere with a heating rate of 10 °C·min−1 between 30 and 600 °C. Bulk density and apparent density were calculated by the ratio of mass to volume with a 50 mL pycnometer based on the standard of SY/T5108-2014. The compressive performance was tested with an electro-hydraulic servo universal testing machine . The breakage ratio was measured by referring to the SY/T5108-2014 standards. For the compressive strength test, 2 g samples were used and the average of three tests was taken. After crushing, the breakage ratio of the particles was calculated by the following formula:Wc is the weight of the crushed sample (g) while W0 is the weight of the sample (g) before crushing.The FTIR spectrum of composites microspheres was obtained with a NICOLET 5700 spectrometer  in the range 400–4000 cmco-MMA), and poly(St-co-MMA)/30 wt.% FA composites. The signal at 1449 cm−1 belongs to the bending vibration of -CH2- deriving from St [−1 was ascribed to C=O stretching vibration, which revealed the existence of C=O groups in MMA [−1 was attributed to Si–O stretching vibration. For the poly(St-co-MMA) copolymer, peaks at 3025 cm−1 and 2919 cm−1 indicate the C–H stretching vibration in St and MMA, respectively. A strong peak at 1728 cm−1 represents the C=O stretching vibration; the C–O stretching vibration in the range of 1000–1200 cm−1 can be assigned to the MMA. Peaks at 700–760 cm−1, corresponding to the C–H bending vibration, belong to the mono-substituted benzene ring, and the peak at 1500 cm−1 belongs to the C=C stretching of the benzene ring in St. For the poly(St-co-MMA)/30 wt.% FA composite, except for the vibration signals of MMA and St, more signal peaks can be observed. The peaks at 3538 cm−1, 1035 cm−1, and 400–600 cm−1 were attributed to the -OH bending of the silicate layers, Si–O stretching, and Si–O–Si bending vibration, respectively [co-MMA)/30 wt.% FA composite. Overall, the FTIR investigations have clearly shown that the FA was successfully incorporated in the polymer matrix [ from St . The peas in MMA . The peaectively ,27. Thesco-MMA) and poly(St-co-MMA)/FA microspheres. In all the samples based on the poly(St-co-MMA), a broad peak appeared between 13°–25°, which reveals that the copolymer materials are mainly amorphous. FA powder is also mainly constituted of an amorphous phase, containing only a small crystalline phase. The characteristic diffraction peak of fly ash appears at 2θ = 26.27° [co-MMA)/FA microspheres. At the same time, with the increase of fly ash content, the characteristic peak strength increased, which indicated that the FA maintained its property in the polymer matrix, and the amorphous structure of the poly(St-co-MMA) did not change [X-ray diffraction (XRD) was used to determine the crystalline phase of the composite microspheres. = 26.27° . A weaket change .co-MMA) and poly(St-co-MMA)/FA microspheres is shown in co-MMA)/FA microspheres gradually roughened, and their interior (The microscope morphology of the prepared poly(St-interior e and surinterior f color gco-MMA) copolymer and poly(St-co-MMA)/FA composites with different FA loadings. As shown in the figure, the onset decomposition temperatures of the poly(St-co-MMA)/FA composites were increased in comparison with that of pure copolymer, which can be ascribed to the existence of the FA in the composite. The pure poly(St-co-MMA) samples had a maximum weight loss temperature of 404 °C, while the maximum weight loss temperature of the 10 wt.%, 30 wt.%, and 50 wt.% FA composites increased to 410 °C, 414 °C, and 417 °C, respectively (co-MMA). With the increase of FA content, the final weight loss of poly(St-co-MMA)/FA microspheres was lower than that of pure poly(St-co-MMA) microspheres; this results illustrates that FA improved the thermal stability of the poly(St-co-MMA) matrix [co-MMA). Thus, the incorporation of FA particles within the poly(St-co-MMA) copolymer matrix could significantly enhance the thermal stability [ectively b. The intability ,31.co-MMA)/FA microspheres with different contents of FA. Both the apparent density and bulk density revealed an increasing trend with the increase of FA content. All of the composite microspheres had an ultra-low density with a bulk density less than 0.7 g/cm3, and the apparent density was between 1 and 1.3 g/cm3, which is close to the density of water. The ultra-low density of the obtained composite microspheres was mainly ascribed to the low density of the polymer matrix  and the low density of micrometer-sized FA (5000 mesh). Thus, the composite particles are lightweight and can achieve a partial monolayer distribution in hydraulic fracturing, which could greatly reduce the proppant concentration per unit area of a fractured well, leading to high fracture conductibility with a low concentration range [co-MMA)/FA microspheres have a potential application as ULW proppants.Bulk density and apparent density are important parameters to evaluate the performance of proppants. on range . These rco-MMA)/FA microspheres have a good crush resistance with 30 wt.% of FA. The reason for this phenomenon is the high interfacial shear strength between the poly(St-co-MMA) matrix and FA that forms from the bonding of cross-links which cover or screen the FA. Consequently, this prevents the expansion of the cracks inside the material, while the expansion of a crack can be transferred due to the good bonding between the poly(St-co-MMA) matrix and FA. Furthermore, the incorporation of the hard FA powders into the poly(St-co-MMA) matrix improves the stiffness of the composites by restricting the movement of the matrix molecular chain [To adapt to the working environment, a proppant also needs to have excellent compressive strength; thus the crushing rate under 52 MPa of pressure was used as the evaluation standard. As shown in ar chain . Howeverco-MMA)/FA was close to 1, which is better than the proppants based on walnut shells (0.65) [co-MMA)/FA ultra-lightweight proppant of this work, all of the aforementioned proppants had a high sphericity (>0.9), ultra-low density, and good compressive strength. The studied proppant has a performance comparable to that of traditional proppants, which allows the proppant to reach the width of the fracture, travel deeper, and improve the conductivity.The results of the sphericity, apparent density, bulk density, and crush resistance tests of various types of proppants are presented in s (0.65) , quartz s (0.65) , and cers (0.65) . Higher s (0.65) . Even ths (0.65)  and inors (0.65) . These as (0.65) ,34,38,39s (0.65) , PMMA/FAs (0.65) , and PS/s (0.65)  compositco-MMA)/FA composite microspheres were successfully synthesized via an in situ suspension polymerization. The properties of the poly(St-co-MMA)/FA were characterized by various techniques and the results demonstrated that FA was successfully introduced into the microspheres of poly(St-co-MMA). All of the obtained composite microspheres had an apparent density in the range of 1–1.3 g/cm3, with a good sphericity >0.9, which is very suitable for hydraulic fracturing. With the incorporation of FA (FA ratio of 30%), the crushing rate decreased to 0.92% at 52 MPa and the maximum weight loss temperature increased from 404 to 414 °C. The studied proppant thus has a comparable performance to that of conventional proppants. Moreover, it also allows improvements in conductivity that are not achieved by conventional proppants because it has an ultra-low density. The preparation approach is facile and controllable, which is suitable for large-scale production. The as-prepared cross-linked poly(St-co-MMA)/FA composite microsphere are a promising material for application as ULW proppants in the shale oil or gas fracturing industry.In summary, poly(St-


The SARS-CoV-2 pandemic has almost stopped all elective surgical treatment throughout the world. As operating room (OR) capacities are reduced everywhere to ensure availability of intensive care capacities, especially low-complex surgical procedures are often postponed. These include totally implantable central-venous access ports which are important for the oncologic treatment of cancer patients.In our study, we investigated the potential of an outpatient surgical centre (OSC) in terms of workflow effectiveness compared to the central operating room complex (COR) of a university hospital using low-complex surgical procedures as an example. Data of 524 consecutive patients who received a Port-a-cath procedure (422 implantations (80.5%) and 102 explantations (19.5%)) in our department between February 2019 and February 2020 were evaluated.p < 0.032). Total OR time (01:08 vs. 01:20 h) and preparation-to-incision time were also shorter in the OSC group .A total of 277 patients were operated in outpatient surgical centre (OSC), and 247 patients received the procedure in the central OR (COR) complex. Grade II and III complications according to the Clavien–Dindo classification occurred in 5.2% (OSC) and 7.3% (COR) of patients. Incision-to-suture time was significantly quicker in the OSC group (36 vs. 42 min., In order to ensure effective OR utilization especially in times of the corona pandemic, the use of smaller decentralized OR units, e.g., outpatient surgical centres, for performing low-complex surgical cases is beneficial. Our study revealed shorter total OR and preparation-to-incision times. As a result of hospital capacity constraints and the swift recognition that COVID‐19 poses an important danger to both patients and healthcare professionals . OperatiCompared to the central OR (COR) complex of a hospital, where workflows are expected to be slower due to the broader range of procedures, length of operations and scope of organization , low-comIn this study, we evaluated data of 524 consecutive patients who received a Port-a-cath procedure in the Department of Surgery, Klinikum rechts der Isar, Technical University Munich (MRI) between February 2019 and February 2020, whereas 277 patients were operated in the outpatient surgery centre (OSC), 244 patients received the procedure in the central OR (COR) complex. Patient assignment to the OSC and COR was done randomly. However, urgent port implantations or explantations during on-call hours were performed in the central operating room. All operations were performed under local anesthesia . Scheduln = 359 patients (85.1%). In n = 63 patients (14.9%), puncture of the subclavian vein (closed strategy) was necessary. No grade IV or V complication according to the Clavien–Dindo classification occurred, especially no pneumo- or hematothorax after vein puncture or postoperative bleeding with the need of surgical revision was observed.A total of 422 patients (80.5%) received a Port-a-cath implantation  due to cancer diagnosis for chemotherapy. In 102 patients (19.5%), the Port-a-cath was removed after chemotherapy was completed or due to complications . For port implantation, cut down technique of the cephalic vein (open strategy) was performed in In the OSC cohort, 229 patients (82.7%) received a Port-a-cath implantation and 48 patients (17.3%) a Port-a-cath explantation.Postoperative complications grade II and III according to the Clavien–Dindo classification occurred in 12 patients with Port-a-cath implantation (5.2%). Of these, 5 (2.2%) were perioperative  and 7 (3.1%) were long-term complications (infection and thrombosis).In the COR cohort, 193 patients (78.1%) received a Port-a-cath implantation and 54 patients (21.9%) a Port-a-cath explantation. Postoperative complications grade II and III occurred in 14 patients with Port-a-cath implantation (7.3%). Of these, 4 (2.1%) were perioperative  and 10 (5.2%) were long-term complications (infection and thrombosis).Table Intraoperative fluoroscopy images  were transferred to the Picture Archiving and Communication System (PACS) via LAN interface using the SAP/KIS compatible worklist of the C-arm software. Conventional postoperative X-ray for exclusion of pneumothorax was performed if the subclavian vein (closed strategy) was punctured.Patient satisfaction was assessed by using a self‐administered questionnaire in the OSC cohort. The scheduling of the surgery date was considered very good by 74% and good by 22.4% of patients. Scheduling of the time slot on the respective OR date was considered very good by 69.6% and good by 25.6% of patients. 3.9% were not satisfied (waiting time to long). The overall patient management was rated very good by 79.8% and good by 3.9% of patients; however, 15.6% did not answer this question. Recommendation for others to have an operation performed in the OSC was made by 75.9% of patients.Primary success rates, tolerability, grade II and III complication rates according to the Clavien–Dindo classification and dose rate of radiation did not differ significantly between the groups. No grade IV and V complication was noted. All patients were seen for follow-up 7–10 days after surgery in the outpatient clinic, patients with Port-a-cath implantation were also seen 3 months postoperatively (first tumor follow-up).p < 0.032). This could be due to the fact, that the procedure was always performed or assisted by the same experienced consultant surgeon in the OSC group, whereas in the COR cohort, different surgeons (varied experience level) performed the procedure.Patient assignment to the OSC and COR was done randomly. However, urgent port implantations or explantations during on-call hours were performed in the central operating room. Mean incision-to-suture time for Port-a-cath explantation was similar between the OSC and COR cohort, however, it was significantly quicker in the OSC group . The time savings can be explained by the fact that the medical and nursing staff in the outpatient surgical centre are better coordinated, communication structures are optimized and the distances in the OSC are generally shorter. The authors assume, that these time savings can be used as best practice for any hospital institution with identical OR structures. Scheduling of the OR date and time slot, as well as rating of the general patient management, was considered very good by 70–80% of patients.Total OR time, including pre- and postprocessing of the patient, was quicker in the OSC group (01:08 vs. 01:20 h) although the results did not differ significantly between the groups. However, the preparation-to-incision time differed significantly (12 min. (OSC) vs. 17 min. (COR), In order to save OR capacity especially in times of the corona pandemic, were predominantly emergency cases and cancer operations should be performed in the central ORs of the hospitals, the use of smaller OR units, such as outpatient surgical centres, for low-complex surgical cases is beneficial. Our study revealed shorter total OR times and a quicker pre- and postprocessing (preparation-to-incision time), enabling a more effective OR utilization with the possibility to perform an increased number of low-complex operations without endangering patient safety.


Acinetobacter baumannii-calcoaceticus (ABC) complex, such as A. baumannii, A. pittii and A. nosocomialis, are a major cause of hospital acquired infections and outbreaks with increasing occurrence of multidrug-resistance. Sulbactam-durlobactam (SUD), a combination of one active β-lactam antibiotic (sulbactam) with a new β-lactamase inhibitor (durlobactam), is currently being tested in a phase 3 clinical trial by Entasis Therapeutics for the treatment of serious infections caused by ABC, including multidrug-resistant strains. At the same time, an ETEST® SUD (sulbactam-durlobactam - MIC range 0.004/4-64/4 µg/mL) has been developed and calibrated versus the broth microdilution reference method (BMD) as described by the Clinical and Laboratory Standards Institute (CLSI). This test is intended to determine the MIC of sulbactam-durlobactam for species of the ABC complex. The aim of this study was to perform a first comparative study of ETEST SUD with the CLSI BMD method on a panel of 263 isolates.Species belonging to the A. baumannii, 29 A. pittii, 30 A. nosocomialis, including 24 SUD-resistant strains, and one CLSI QC strain. BMD was performed using the 2021 CLSI guidelines. ETEST SUD was evaluated using the standard ETEST procedure for Acinetobacter spp. . For each method, the MIC was read at complete inhibition of visible growth. To determine category agreement (CA) and error rates, the sulbactam-durlobactam provisional breakpoint of 4 µg/mL was applied.The panel consisted of 204 The QC strain MICs were in the expected range with reproducible results. The essential MIC agreement  was 97.7% without any tendency to over- or underestimate the MIC when compared to BMD. The CA was 98.5%. Two Very Major Errors, both within the EA, and two Major Errors, one within the EA, were observed.In this study, the ETEST SUD was found to be equivalent to the CLSI reference method. MIC end points were easy to read. With a 15-dilution range and simplicity of use, ETEST SUD could represent a valuable tool for MIC determination and could be an alternative to BMD.For Research Use Only. The performance characteristics of this product have not been established yet.All Authors: No reported disclosures


Thyroid cancer is one of the most common endocrine malignancies worldwide, and papillary thyroid cancer (PTC) is the most common pathologic type of thyroid cancer. SQSTM1/p62 activity mediates different biological functions. This study aimed to investigate the effect of SQSTM1/p62, a multifunctional receptor, on biological function and autophagy characteristics in the human PTC cell line TPC-1.in vitro. TPC-1 and p62-KO-TPC-1 human PTC cell lines in the logarithmic growth phase were subcutaneously implanted into BALB/c nude mice to verify their proliferation effect in vivo. Furthermore, western blotting and immunohistochemistry (IHC) were used to detect the expression of AKT/AMPK/mTOR signaling pathway-related proteins.A total of 105 primary PTC samples and matched adjacent normal thyroid tissue samples were obtained to evaluate the expression of p62 in clinical patients. A similar p62 expression pattern was found in PTC cell lines and normal human thyroid follicular epithelial cells. To evaluate the effect of SQSTM1/p62 on TPC-1 cells, we constructed the p62 knockout cell line p62-KO-TPC-1. Cell proliferation, cell cycle, and cell apoptosis were analyzed by colony formation tests, Cell Counting Kit-8 (CCK-8) assays and flow cytometry in vitro, p62 deficiency could decrease the number of colonies, inhibit cell growth and the cell cycle, and induce apoptosis. Tumor xenograft experiments in BALB/c nude mice corroborated these findings. Moreover, the molecular mechanism was explored by western blotting, and we found that the AMPK/AKT/mTOR pathway was involved.Overall, p62 expression was higher in tumor tissues than in normal tissues in 73 of 105 PTC patients (69.5%). The expression level of p62 in the PTC cell line was higher than that in the normal thyroid cell line. Our data indicated that via the AMPK/AKT/mTOR pathway and could be used as a potential therapeutic approach for PTC.The results indicate that p62 might mediate cell autophagy and apoptosis in TPC-1 cells  Epithelial follicular-cell-derived thyroid cancer is one of the most common malignancies of the endocrine system worldwide, and its incidence has sharply in recent years –7. InterThe occurrence and development of thyroid tumors is a complex process involving multiple genes, including cancer suppressor genes, cancer promoting genes and many regulator genes , 16. It The sequestosome 1 gene (SQSTM1), also named p62, is located in 5q35, and it is an adaptor protein in the ubiquitination system as well as a cargo protein receptor in selective autophagy that plays an important role in regulating intracellular protein degradation . NeverthAn increasing volume of evidence indicates that p62 participates in many signal transduction pathways, including insulin, Keap1-Nrf2, ERK, and p38/MAPK signaling pathways . TherefoA total of 105 primary PTC samples and matched adjacent normal thyroid tissue samples were obtained at the time of initial surgery and snap-frozen immediately after tumor removal. Inclusion criteria: all patients had not received radiotherapy, chemotherapy or other treatment measures before surgery; all patients underwent thyroid cancer surgery for the first time; radical operation of thyroid carcinoma was performed in all cases; pathological results were double-blind review by pathologists and diagnosed as papillary thyroid carcinoma. Exclusion criteria: the patients had a history of other malignant tumors; accompanied by cardiac insufficiency, liver and kidney failure and other important organs of serious lesions; malignant infection.The study was approved by the Ethics Committee of Zhengzhou University, and human tissues were obtained with informed written consent from the patients. All tissue samples were reviewed by an endocrine pathologist to confirm the diagnosis. PTC samples estimated to contain more than 80% tumor cells were used. This study was approved by the ethical standards of the institutional ethics committee, and informed consent was obtained from all patients (No. 2019-KY-314).From March 2019 to September 2019, 105 patients with pathologically proven PTC were included in this study. All patients were aged 24 to 62 years .2 at 37°C. The following chemicals were used: rapamycin  and bafilomycin A1 .The human PTC cell line TPC-1 was kindly provided by Dr Ye Lei  and the normal human thyroid follicular epithelial cell line Nthy-ori3-1 was purchased from Shanghai Cell Biochemical Institute . All the cell lines were cultured in RPMI-1640 medium  with 10% fetal bovine serum (FBS)  and incubated in a humidified atmosphere of 5% COTo investigate the effect of p62 on TPC-1 cells, the CRISPR/Cas9 technique was used to construct the p62 knockout cell model p62-KO-TPC-1. The p62 knockout colony was generated by CRISPR/Cas9-mediated genome editing. The sgRNA was designed and synthesized to specifically target p62. The sgRNA guide sequence was 5′-TGGCTCCGGAAGGTGAAACA-3′. The synthesized and validated Lenti-CAS9-sgRNA-Puromycin vector was transferred into TPC-1 cells. Single cells were expanded to obtain individual clones that were lysed and quantified. The absence of p62 was verified by western blotting and RT-qPCR.A Cell Counting Kit-8 assay  was first performed. Tumor cells were cultured in 96-well plates at a density of 1000 cells per well. The next day, 10 μl CCK-8 reagent was added to each well. After a further incubation of 4 hours at 37°C, the cell proliferation rate was assessed by measuring the absorbance at 450 nm with the Universal Microplate Reader.Colony formation assays were subsequently conducted. A total of 500 cells were plated in 6-cm dishes for 2 weeks. Then, the cells were fixed with methanol for 20 minutes and stained with crystal violet. The colonies were counted.Data are shown from three independent experiments performed in triplicate wells.6 cells were collected and injected subcutaneously into the flank regions of 4-week-old female BALB/c nude mice . The tumor volume was measured at the indicated time after inoculation. After 3 weeks, the tumor-bearing mice were sacrificed, and the average weight of the tumor tissues was measured. Sections of tumor tissues were detected by immunohistochemistry (IHC). The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. 2019-KY-314).A total of 2×10® reagent , and the PrimeScript RT reagent Kit with gDNA Eraser  was used to remove genomic DNA and synthesize complementary DNA (cDNA). Quantitative real-time PCR was performed in triplicate using the RT-qPCR mRNA SYBR Green Detection kit . The threshold cycle (Ct) value was recorded, and changes in mRNA levels were determined by the 2-ΔΔCt method using GAPDH for internal crossing normalization.The expression of the p62 gene was detected by RT-qPCR. The primers for the p62 and GAPDH genes were synthesized by Shanghai Sangon Biotech, and the sequences were as follows: p62 forward, 5’-AGGCGCACTACCGCGAT-3’ and reverse, 5’-CGTCACTGGAAAAGGCAACC-3’; GAPDH forward, 5’-GGTCGTATTGGGCGCCTGGTC-3’ and reverse, 5’-TGACGGTGCCATGGAATTTGCCA-3’. Total RNA was extracted from primary PTC samples and matched adjacent normal thyroid tissue samples, TPC-1 and p62-KO-TPC-1 cells (continuously cultured in puromycin-free medium for ≥4 weeks) using TRIzolThe Annexin V-FITC Apoptosis Detection Kit (KeyGen BioTECH: KGA105-KGA108) was used to assay the apoptosis of TPC-1 and p62-KO-TPC-1 cell lines. The apoptotic cells labeled with annexin V were assessed by a flow cytometer . Data were obtained from three independent experiments.The Cell Cycle Detection Kit (KeyGen BioTECH: KGA511-KGA512) was used to assay the cell cycle of TPC-1 and p62-KO-TPC-1 cells. The tumor cells labeled with PI/RNase A fluid were detected by a flow cytometer , and the data were analyzed using Multicycle-DNA Cell Cycle Analysed Software. Data were obtained from three independent experiments.After culturing in RPMI-1640 medium with 10% FBS, TPC-1 and p62-KO-TPC-1 cells were lysed in R IPA lysis buffer  containing 1% Protease Inhibitor Cocktail . Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane . After blocking with 5% skimmed milk, the PVDF membrane was then incubated with the following antibodies: p62 , AKT , P-AKT , AMPK , Bax , Bcl-2 , mTOR , LC3A , actin , and GAPDH .The results are expressed as the mean ± SD for at least three separate experiments. Statistical evaluation was performed using Prism (GraphPad) software. Statistical significance was assessed using a two-tailed unpaired Student’s t-test, Chi-square test and Fisher’s exact test. P < 0.05 was considered to indicate a statistically significant result.p=0.0066) . The results showed that the p62 expression level was significantly increased in PTC tissues compared to normal thyroid tissues. As shown in =0.0066) Table 1.in vitro and in a normal human thyroid follicular epithelial cell line (Nthy-ori3-1). Similar to the results obtained using tissue samples, p62 expression was higher in the PTC cell line than in normal human thyroid follicular epithelial cells  is known as an energy sensor that regulates cellular metabolism by mediating the insulin pathway, and its phosphorylation is triggered by glucose stress or hypoxia in the tumor microenvironment . However pathway , while A pathway . ConsideIn summary, LC3-II expression was increased after p62 knockout, but no significant accumulation was observed after Bafilomycin A1 application, suggesting that autophagy was inhibited. The western blotting results of related pathway proteins showed that p62KO promoted the phosphorylation of AKT, inhibited the expression of AMPK and caused the activation of mTOR, which made it difficult for autophagy to be started and led to autophagy inhibition, indicating that p62 played a role in inducing autophagy in papillary thyroid carcinoma. Enhancement of autophagy leads to enhanced decomposition of intracellular metabolic wastes, and the protein raw materials produced can generate proteins required by cells through a series of synthesis effects, maintain intracellular homeostasis, and help tumor cells to continue to survive under harsh conditions . TherefoThis study revealed the tumor-promoting effect of p62 in PTC by AKT/AMPK/mTOR pathway, contributing to the basic research on the occurrence and development of PTC, and further suggesting that p62 or p62 related molecule may be a diagnostic and therapeutic target for PTC.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.All authors have made substantial contributions to the work, including editing and writing assistance, reported in the manuscript. FY, RM, CL, and LZ collaborated in the study conception and design. Cases and blocks were selected from the First Affiliated Hospital of Zhengzhou University by KF and MW. Data collection and analysis were performed by FY. The first draft of the manuscript was written by FY, and all authors commented on previous versions of the manuscript. The entire experimental process was supervised and guided by DY. All authors contributed to the article and approved the submitted version.The present study was supported by grants from the University Scientific and Technological Innovation Team Project of Henan Province (19IRTSTHN002); The Thousand Talents Science and Technology Innovation Leading Talents Subsidy Project of Central Plains (194200510011); Major Scientific Research Projects of Traditional Chinese Medicine in Henan Province (No.20-21ZYZD14); Cultivation of Young and Middle-aged Health Science and Technology Innovation Leading Talents in Henan Province (YXKC2020015).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IntroductionA cytokine storm is an important cause of morbidity and mortality in patients with coronavirus disease 2019 (COVID-19). The objective of the study was to determine the prognostic significance of pro-inflammatory cytokines with the overall final outcome of patients with COVID-19.MethodsWe conducted a retrospective study of 142 patients admitted with COVID-19 in the Department of Medicine at All India Institute of Medical Sciences, New Delhi, from May 2021 to June 2021. We obtained their demographic, clinical, and biochemical characteristics at baseline and 48-72 hours prior to the terminal event . The data were analyzed to determine the prognostic significance of these markers on the final outcome.ResultsHigher levels of inflammatory markers were associated with a worse final outcome  p-value <0.001, interleukin 6 (IL-6) p-value 0.007, procalcitonin p-value 0.005, and lactic acid p-value 0.004). Optimal probability cut-offs for these markers for predicting mortality were: ferritin: 963 ng/mL , CRP: 66.3 mg/L , IL-6: 46.2 pg/mL , procalcitonin: 0.3ng/mL , lactic acid: 1.5 mg/dL . Multivariate logistic regression analysis was done, which showed that pre-terminal event CRP was associated with a statistically significant higher risk of mortality .ConclusionInflammatory markers have a prognostic significance in patients with COVID-19, with higher levels being associated with worse outcomes. Coronavirus disease 2019 (COVID-19) is a raging pandemic that has affected millions of people worldwide. While most of the cases have an asymptomatic or mild course, a significant number of patients develop severe disease, often associated with a hyperinflammatory state called a cytokine storm .Cytokine storms manifest clinically with rapid clinical deterioration characterized by coagulopathy, hypotension, and multiple end-organ dysfunction . SeveralAnti-inflammatory therapies directed at reducing the levels of these cytokines may be associated with better clinical outcomes. Therefore, we conducted a retrospective study to determine the correlation of the trend of pro-inflammatory cytokines, including ferritin, C-reactive protein (CRP), interleukin 6 (IL-6), procalcitonin, and lactic acid with the overall outcome of patients with COVID-19.Study designThis was a retrospective observational study of 142 patients admitted with COVID-19 in the Department of Medicine at All India Institute of Medical Sciences, New Delhi, from May 2021 to June 2021. The study included all patients who were 18 years or older, with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection confirmed by real-time reverse-transcription-polymerase chain reaction (RT-PCR) assay of nasal, pharyngeal, or lower respiratory tract samples. The study protocol was approved by the Ethics Committee of the All India Institute of Medical Sciences, New Delhi, with the IEC number: IEC-295/17.04.2020, RP-37/2020. We extracted the medical records and charts of each patient and reviewed all the data. Since the study was a retrospective study and did not hamper patients’ privacy, informed consent was waived.Clinical and biological dataIn this study, medical history, demographic characteristics, comorbidities, illness severity on admission, and laboratory findings were collected.Laboratory investigations, including blood counts and liver and kidney function tests, were obtained at baseline. Serum ferritin, CRP, IL-6, procalcitonin, and lactic acid were collected at baseline and 48-72 hours prior to the final outcome of patients, and their change from baseline values was also noted. The primary outcome was patient survival during hospitalization. The final outcome of these admitted patients was categorized as either survivor or non-survivor.Statistical analysisStatistical analysis was conducted to determine the prognostic significance of these markers on the final outcome  using StataCorp. 2019 . All categorical variables were expressed as frequency and percentage. All continuous variables were expressed as mean and standard deviation or median and range. Parameters of the individuals among survivors and non-survivors were compared using the chi-square test or Fisher’s exact test for categorical variables, independent t-test for continuous variables following normal distribution, and Wilcoxon rank-sum test for parameters following non-normal distribution. Receiver operating characteristic (ROC) curves were used to obtain the optimal probability cut-off to predict the final outcome of these markers. A p-value of <0.05 was considered significant.Demographic and clinical featuresA total of 142 patients were included in the study and their demographic characteristics and clinical features are described in Table Among the non-survivors, hypertension was present in 31 (56.36%) patients while amongst the survivors, it was present in 27 (31.03%) patients, and this difference amongst the two groups was statistically significant (p = 0.003).Laboratory investigationsThe laboratory investigations of survivors and non-survivors are described in Table Inflammatory biomarkersIn the study, the values of inflammatory markers among non-survivors and survivors and compared their median values among the two groups are shown in Table The median ferritin level at baseline among non-survivors was 1009 ng/mL  as compared to 659 ng/ml  among survivors and this difference was statistically significant . The median ferritin level prior to the terminal event  among non-survivors was 1395 ng/mL  as compared to 600.5 ng/mL  among survivors and this difference was statistically significant . The median value of change in ferritin level from baseline to the pre-terminal event among non-survivors was 44 ng/mL , whereas, among survivors, it was -0.4 ng/mL . However, this difference was not statistically significant (p = 0.07).Hence, higher ferritin levels prior to the terminal event  and at baseline, were associated with a greater probability of an adverse outcome . Ferritin levels prior to the terminal event were more strongly correlated with death than ferritin levels at baseline. The change in ferritin values during the course of hospital admission, however, was not associated with the final outcome of patients.The optimal probability cut-off for ferritin for predicting the final outcome prior to the terminal event was calculated using the ROC curve and came out to be 963 ng/mL, with a sensitivity of 67.35% and specificity of 67.50% in predicting the final outcome, that is, survival or death of patients Figure .The median CRP level at baseline among non-survivors was 102.45 mg/L  as compared to 37.94 mg/L  among survivors and this difference was statistically significant (p <0.001). The median CRP level prior to the terminal event  among non-survivors was 116.8 mg/L  as compared to 14.38 mg/L  and this difference was statistically significant (p <0.001). The median value of change in CRP level from baseline to the pre-terminal event among non-survivors was 0  and 0  and the p-value was statistically insignificant (p = 0.13). Hence, higher CRP levels prior to the terminal event and those obtained at baseline were associated with a greater probability of death . The association was stronger for CRP levels prior to the terminal event than CRP levels at admission. The change in CRP values during the course of hospital admission was not associated with the final outcome of the patients.Optimal probability cut-off for CRP prior to the terminal event for predicting the final outcome was calculated using the ROC curve and came out to be 66.7 mg/L with a sensitivity of 78.43% and specificity of 74.12% in predicting the final outcome, that is, survival or death of patients Figure .The median IL-6 level among non-survivors was 53.84 pg/mL  as compared to 37.94 pg/mL  among survivors. Hence, higher IL-6 levels were significantly associated with an adverse final outcome (p = 0.007).The optimal probability cut-off for IL-6 was calculated using the ROC curve and came out to be 46.2 pg/mL with a sensitivity of 59.26% and a specificity of 59.57% in predicting the final outcome, that is, survival or death of patients Figure .The median procalcitonin level among non-survivors was 0.73 ng/mL  while the median procalcitonin level amongst survivors was 0.15 ng/mL . Hence, higher procalcitonin levels were significantly associated with an adverse outcome (p = 0.005).Optimal probability cut-off for procalcitonin for predicting the final outcome was calculated using the ROC curve and came out to be 0.3 ng/mL with a sensitivity of 65.38% and specificity of 66.67% in predicting the final outcome, that is, survival or death of patients Figure .The median lactic acid level among non-survivors was 1.70 mmol/L  as compared to 1.30 mmol/L . Hence, higher lactic acid values were significantly associated with a worse final outcome (p = 0.004).The optimal probability cut-off for lactic acid for predicting the final outcome was calculated using the ROC curve and came out to be 1.5 mmol/L with a sensitivity of 59.26% and specificity of 58.57% in predicting the final outcome, that is, the survival or death of patients Figure .Multivariate logistic regression analysis was done for the pre-terminal event CRP, pre-terminal event ferritin, lactic acid, age, and hypertension. A total of 113 observations were available for the multivariate analysis. However, due to the small sample size for patients included in the multivariate analysis, only pre-terminal event CRP was associated with a statistically significant higher risk of mortality . No other variables were associated with a statistically significant higher risk of mortality in COVID-19 disease as shown in Table We conducted a retrospective analysis of 142 COVID patients. We found that inflammatory markers, including ferritin, CRP, IL-6, procalcitonin, and lactic acid, were significantly raised in non-survivors as compared to survivors. Several studies have been conducted to ascertain the relationship between these markers and the overall outcome of patients with COVID-19 disease. Inflammatory markers have been reported to be predictors of mortality in patients with COVID-19 disease . A definOur study showed that high serum ferritin values prior to the terminal event  were more significantly associated with death as the final outcome than hyperferritinemia at baseline or the absolute change in serum ferritin values during the course of hospital admission with respect to the baseline values. High serum ferritin values at the time of admission have been independently associated with a severe disease course . HyperfeSimilarly, it was observed in our study that higher CRP levels prior to the terminal event  were more significantly associated with an adverse final outcome than the CRP values obtained at baseline and the absolute change in CRP values from baseline during the course of hospital admission. Earlier reports have also indicated that CRP levels at the time of admission and prior to discharge or death are markers of poor prognosis in patients with COVID-19 . Higher We observed that IL-6 was significantly higher amongst non-survivors as compared to survivors in our study group. These findings reiterate the fact that COVID-19 is a hyperinflammatory state associated with an increase in pro-inflammatory cytokines and raised inflammatory markers are associated with a severe disease course and an adverse final outcome as suggested by earlier studies -16.In our study, we observed that higher procalcitonin levels were associated with a worse prognosis. These findings are in concordance with earlier studies reported in the literature . ElevateWe also observed that elevated lactic acid values were associated with a worse overall outcome of patients. This has also been suggested by other studies reported in the literature . Lactic All the variables with a significant p-value on univariate analysis were then analyzed using the Cox regression model. However, only pre-terminal event CRP was significantly associated with a worse overall outcome. This may be attributed to the very small sample size available for multivariate analysis.The limitations of our study included the small sample size and the retrospective nature of our study. The effect of various therapies, including anti-inflammatory drugs, and other supportive therapies, such as antibiotics, vasopressors, etc., also could not be studied, as this was a retrospective study and, hence, precluded the randomization of study participants to different treatment protocols.Inflammatory markers, including ferritin, CRP, IL-6, procalcitonin, and lactic acid, have prognostic significance in patients with COVID-19 disease. Higher levels of these inflammatory markers, particularly later during the course of hospital admission, are associated with a higher likelihood of non-survival in these patients. An increase in these inflammatory markers is an early indicator of cytokine storm in these patients, which is a harbinger of a relatively poor prognosis and is associated with higher mortality in patients with COVID-19. This will help clinicians in the early identification of high-risk patients. Therefore, measuring the levels of these inflammatory markers early in the disease helps in prognosticating patient outcomes. This, in turn, helps in tailoring early, appropriate, efficient, and effective treatment of patients, which will lead to a significant reduction in mortality related to COVID-19.


Logistic regression analysis revealed that the expression of miR-200c-3p and systemic arterial hypertension are factors independently associated with patients in group IV (p<0.0001). Our results suggest that miR-200c-3p is a predictor of severity independent of COVID-19 risk factors, which could represent a way of screening patients affected by SARS-CoV-2.COVID-19 represents a public health emergency, whose mechanism of which is not fully understood. It is speculated that microRNAs may play a crucial role in host cells after infection by SARS-CoV-2. Thus, our study aimed to analyze the expression of miR-200c-3p in saliva samples from patients with COVID-19. One handred eleven samples from patients with COVID-19 were divided into 4 groups. Group I: 39 patients negative for Covid-19; Group II: 37 positive and symptomatic patients, with no indication of hospitalization; Group III: 21 patients with respiratory disorders ; Group IV: 14 patients with severe conditions (oxygen therapy). The expression levels of miR-200c-3p were determined using qPCR. We found greater expression of miR-200c-3p in patients in group IV ( MicroRNAs (miRNAs) are small endogenous molecules that do not encode proteins that bind mRNA targets of several genes and can control their expression at the posttranscriptional level . miRNAs Viral infections regulated by the action of miRNA have already been described in several hosts, both in infections caused by DNA and RNA viruses . With thCOVID-19 (Coronavirus Disease-2019) represents a public health emergency . WorldwiStudies with microRNAs can contribute not only to the understanding of the virus-host interaction but also to stratify the different degrees of severity presented by COVID-19. In this sense, miR-200c-3p, which is associated with viral infections, including influenza A , presentPatients: This quantitative longitudinal analytical study was carried out in two hospitals in the city of São Paulo - Hospital Moriah and Hospital das Clinicas of the Faculty of Medicine of the University of São Paulo (HCFMUSP), Brazil from June to October 2020. The sample consisted of 111 patients who donated saliva samples. All patients were tested for SARs-CoV-2 using the qPCR  technique. Patients were divided into 4 groups. Group I was composed of 39 patients with a negative test for Covid-19. Group II was composed of 37 patients with symptomatic clinical conditions and with no indication of hospitalization (absence of respiratory dysfunction). Group III was composed of 21 symptomatic patients who had respiratory disorders; and therefore, these patients needed hospitalization. Group IV, consisted of 14 patients with severe conditions, requiring oxygen therapy and, consequently, were admitted to the Intensive Care Unit of HCFMUSP. Clinical-demographic characteristics of patients are shown in Procedures: To measure the levels of microRNA expression, all patients were instructed to produce a saliva sample at the time of the COVID-19 test collection. The patients remained for at least 1 minute without swallowing and at the end, they spit all the saliva stored in the mouth in the collection tube. In patients admitted to the ICU on oxygen therapy, a sublingual smear was performed with the aid of a swab and subsequently, it was immersed in a collecting tube containing 2 mL of saline solution. All collections were guided and supervised by the nursing team. The samples were stored at -20°C until extractions were performed. Extraction of microRNA and quantitative real-time polymerase chain reaction: To extract the total RNAs, the EasyExtract DNA-RNA kit (Interprise®) was used, following the manufacturer's recommendations. After extraction, the miRNA cDNA was generated using the TaqMan miRNA reverse transcription kit . The reactions were incubated at 16ºC for 30 min, 42ºC for 30 min, and 85ºC for 30 min. The expression levels of hsa-miR-200c-3p (478351_mir) were analyzed by qPCR using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The target sequence was amplified in a 10 µL reaction mixture containing 2 µL of HOT FIREPol Probe Universal qPCR Mix (Solis BioDyne), 0.5 µL of TaqMan miRNA for miR-200c, 1 µL of cDNA, and 6.5 µL of DNase-free water. The conditions of the PCR cycles were 2 min at 50ºC, 10 min at 95ºC and then 45 cycles of 15 s at 95ºC, and 1 min at 60ºC. The normalized relative expression was calculated by thresholding the Ct values of miR-200c with the Ct value of miR-191  [ibrator) . The datStatistical analysis: We used the Kolmogorov-Smirnov test to assess the normality of our data. When the data were parametric, we use Student's t-test to compare the levels of expression between groups. When the data were nonparametric we use the Mann-Whitney test to compare the levels of expression between groups. To analyze more than two groups we use the ordinary one-way ANOVA test for the hypothesis test. Contingency analysis was performed using the Chi-squared test. To assess independently associated correlations between group IV and the miR-200c, we performed a logistic regression. The graphs and the statistical analysis were performed using GraphPad Prism 8 software. The result was considered significant when p≤0.05. The data are detailed in Supplementary 1, 2, and 3. p<0.0001) (p=0.013)  (p=0.0002) (p=0.046)  . It was p=0.013) . Regardi(p=0.208 . When pactively) . Conside=0.0002) . In malep=0.046) .p<0.0001 (p<0.0001). Interestingly, only patients belonging to groups III and IV had both hypertension and diabetes, and again, group IV has a higher percentage of patients with both comorbidities, being statistically significant when compared to the other groups 35.71% (n=5), p<0.0001. A logistic regression analysis was performed to identify variables independently associated with COVID-19. In this result, we found that the expression of miR-200c-3p and SAH are factors independently associated with patients with severe COVID-19 (Group IV), p<0.0001  71.43% (n=10), p<0.0001 . Similarp<0.0001 .Considering that there is still no specific treatment for COVID-19, the identification of molecules that can contribute to better patient management can be a useful tool in facing the pandemic . MicroRNBuggele et al. found that in influenza A, miR-200c-3p increases its level of expression progressively after infection . We beliLu et al., demonstrated through a luciferase assay that miR-200c is a direct target of ACE2 in human cardiomyocyte cells and suggested that this miR could be used as a preventive treatment by decreasing the expression levels of ACE2 [Advanced age seems to be one of the factors with the worst prognosis in COVID-19 . When coGender disparity was already evident in COVID-19, where lethality has been more prominent in males (17.7%) versus females (10.7%) . MicroRNSimilar to other relevant data presented in the literature, pre-existing comorbidities such as diabetes and hypertension do seem to have an unfavorable role in patients who oppose COVID-19. Corroborating these data, our study demonstrates that a higher percentage of patients who had diabetes and hypertension were higher in the two groups that had unfavorable characteristics, group III and IV. Notably, after logistic regression analysis, it was confirmed that in severe cases, the expression of miR-200c-3p is independently associated with those patients with hypertension. The results found in our study show that miR-200c-3p has differential expression in patients with more aggressive COVID-19 conditions, as well as revealing important associations with age.The use of saliva as a biomaterial for the diagnosis of COVID-19 has been widespread since April 2020 after its release by the FDA. However, the use of this fluid presents some biases pointed out by the scientific community, mainly due to the lack of standardization of protocols, such as optimization of collection, transport, storage of samples, and test methods. However, sensitivity for diagnosis from saliva can vary from 91 to 98% . AlthougAnother important point in the rain of information about the SARs-CoV-2 pandemic is the increase in the number of articles found on pre-publication platforms (pre-prints). Quick access to information brings knowledge that can positively change the clinical outcome of countless patients , however Finally, these results, unique in the literature, may contribute to the use of this microRNA as a possible prognostic marker in the screening of patients. This work opens perspectives for the use of these important molecules in clinical practice, since the quantification of miRNAs is similar to one of the diagnostic techniques of COVID-19, qPCR.The authors declare that they have no conflict of interests.